Metabolite Production in Alkanna tinctoria Links Plant Development with the Recruitment of Individual Members of Microbiome Thriving at the Root-Soil Interface

ABSTRACT Plants are naturally associated with diverse microbial communities, which play significant roles in plant performance, such as growth promotion or fending off pathogens. The roots of Alkanna tinctoria L. are rich in naphthoquinones, particularly the medicinally used enantiomers alkannin and shikonin and their derivatives. Former studies already have shown that microorganisms may modulate plant metabolism. To further investigate the potential interaction between A. tinctoria and associated microorganisms, we performed a greenhouse experiment in which A. tinctoria plants were grown in the presence of three distinct soil microbiomes. At four defined plant developmental stages, we made an in-depth assessment of bacterial and fungal root-associated microbiomes as well as all extracted primary and secondary metabolite content of root material. Our results showed that the plant developmental stage was the most important driver influencing the plant metabolite content, revealing peak contents of alkannin/shikonin derivatives at the fruiting stage. Plant root microbial diversity was influenced both by bulk soil origin and to a small extent by the developmental stage. The performed correlation analyses and cooccurrence networks on the measured metabolite content and the abundance of individual bacterial and fungal taxa suggested a dynamic and at times positive or negative relationship between root-associated microorganisms and root metabolism. In particular, the bacterial genera Labrys and Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium as well as four species of the fungal genus Penicillium were found to be positively correlated with higher content of alkannins. IMPORTANCE Previous studies have shown that individual, isolated microorganisms may influence secondary metabolism of plants and induce or stimulate the production of medicinally relevant secondary metabolism. Here, we analyzed the microbiome-metabolome linkage of the medicinal plant Alkanna tinctoria, which is known to produce valuable compounds, particularly the naphthoquinones alkannin and shikonin and their derivatives. A detailed bacterial and fungal microbiome and metabolome analysis of A. tinctoria roots revealed that the plant developmental stage influenced root metabolite production, whereas soil inoculants from three different geographical origins in which plants were grown shaped root-associated microbiota. Metabolomes of plant roots of the same developmental stage across different soils were highly similar, pinpointing to plant maturity as the primary driver of secondary metabolite production. Correlation and network analyses identified bacterial and fungal taxa showing a positive relationship between root-associated microorganisms and root metabolism. In particular, the bacterial genera Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium and Labrys as well as the fungal species of genus Penicillium were found to be positively correlated with higher content of alkannins.

Dear Dr. Angela Sessitsch: Thank you for submitting your manuscript to mSystems.
The processing of this revision has taken longer than anticipated as I was unable to recruit the three initial Reviewers who commented on your manuscript. Furthermore, the Reviewers provided me with contrasting opinions and I had to act as a tiebreaker.
First of all, I'd like to praise the effort the authors put in the revised version which I consider a significant improvement compared to the original submission. While the concern of Reviewer #1 is understandable, I am of the opinion that the experimental data presented and analysed in the revised version of the manuscript will intercept the interest of a broad scientific community and will likely fuel follow-up investigations.
I am therefore inclined to consider the revised version for publication. There are, however, a few points I'd like the authors to consider (besides the specific comments of the Reviewers below) #1) I think you can be more explicit in the title and impact statement. For instance you could go with something along the lines 'Metabolite production in Alkanna tintoria is driven by plant's development and is sufficiently resilient to microbiome variation across soils' or 'Metabolite production in Alkanna tintoria links plant development with the recruitment of individual members of microbiome thriving at the root-soil interface'. Likewise, L. 61-62 how? I mean what kind of implications you have in mind? Target inoculation with rhizobia to trigger a differential metabolite production? #2) L297-299. I believe is R>0.3. Regardless, I'd justify the rationale for this choice as it is different from what reported in 284-286.
#3) L326-28. The soils used display very similar pH, it could be that in more acidic soil you may have different responses in the plants. I'd include a disclaimer here. #4) L334-335: that's your closing for the importance statement! #5) L386-388. 'Data available on request' is inadmissible. Either provide access to the data or remove the sentence altogether. #6) I found Figure S5 more informative than Figure 7: have you considered swapping them? #End from my side Thank you for the privilege of reviewing your work. Below you will find instructions from the mSystems editorial office and comments generated during the review.

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The ASM Journals program strives for constant improvement in our submission and publication process. Please tell us how we can improve your experience by taking this quick Author Survey. The manuscript has been extensively edited and improved with respect to the previous version. Howver, the main point arisen by this reviewer was not satistified, since it is inherent to the experimental design. In particular the result description and comments are based on correlation analyses, well done, but which do not provide causation and/or mechanistic intepretation of the interpplay between metabolites and microbiota.
Reviewer #5 (Comments for the Author): The manuscript by Csorba et al. provide interesting evidence that growth-stage-dependent variation in specialized metabolite content co-occurs with changes in microbiome composition, suggesting a potential causal role of plant metabolites in modulating the microbiome (or vice versa). While the evidence for this is purely correlational, the study generates useful hypotheses for future study.
The previous comments of the reviewers have all been adequately addressed, and the manuscript has been significantly improved.
I just have a few small notes: • There are some errors in the references in the submitted PDF: 'Error! Reference source not found.' • The various figures seem to be using different fonts. I suggest making this consistent between the figures (all sans serif for example?). Italization of genera is also missing in figure 3, and (non-)capitalization of labels is not consistent across figures.

Reviewer #5
The manuscript by Csorba et al. provide interesting evidence that growth-stage-dependent variation in specialized metabolite content co-occurs with changes in microbiome composition, suggesting a potential causal role of plant metabolites in modulating the microbiome (or vice versa). While the evidence for this is purely correlational, the study generates useful hypotheses for future study.
The previous comments of the reviewers have all been adequately addressed, and the manuscript has been significantly improved.
I just have a few small notes: • There are some errors in the references in the submitted PDF: 'Error! Reference source not found.'

Response:
The errors were due to the cross-referencing of figures and tables in the original document. We removed these in the updated version.
• The various figures seem to be using different fonts. I suggest making this consistent between the figures (all sans serif for example?). Italization of genera is also missing in figure 3, and (non-)capitalization of labels is not consistent across figures.

Response:
We corrected the italization of the genera wherever we could and was needed on the figures, we also replotted everything so the fonts are uniform and have a sans-serif style. Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. For your reference, ASM Journals' address is given below. Before it can be scheduled for publication, your manuscript will be checked by the mSystems production staff to make sure that all elements meet the technical requirements for publication. They will contact you if anything needs to be revised before copyediting and production can begin. Otherwise, you will be notified when your proofs are ready to be viewed.
Please note that the last three lines of the Summary and Impact statement are identical. I believe this is a typo (as the authors agreed on a different conclusion for the impact statement): please rectify it at the proof stage.
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